A novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles.
A novel protein purification method was developed using microbial polyhydroxyalkanoates (PHA) granule-associated protein phasin, a pH-inducible self-cleaving intein and PHA nanoparticles. Genes for the target proteins to be produced and purified were fused to genes of intein and phasin, the genes were jointly over-expressed in vivo, such as in E. coli cells in this study. The fused proteins containing target protein, intein and phasin produced by the recombinant E. coli were released together with all other E. coli proteins via a bacterial lysis process. They were then adsorbed in vitro to the surfaces of the hydrophobic polymer nanoparticles incubated with the cell lysates. The nanoparticles attached with the fused proteins were concentrated via centrifugation. Then, the reasonably purified target protein was released by self-cleavage of intein and separated with nanoparticles by a simple centrifugation process. Using this system, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and beta-galactosidase were successfully purified in their active forms with reasonable yields, respectively, demonstrating the effectiveness and reliability of this purification system. This method allows the production and purification of high value added proteins in a continuous way with low cost.
Wang
Zhihui
Z
Multidisciplinary Research Center, Shantou University, Shantou 515063, Guangdong, China.
Wu
Hongning
H
Chen
Jiong
J
Zhang
Jing
J
Yao
Yongchao
Y
Chen
Guo-Qiang
GQ
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D047668:Inteins; D058619:Maltose-Binding Proteins; D053758:Nanoparticles / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D002264:Carboxylic Acids; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D058619:Maltose-Binding Proteins; C118828:PHAP protein, Bacteria; D011108:Polymers; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase